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Regulation of HPV16 Early Gene Expression

Zheng, Yunji LU (2021) In Lund University, Faculty of Medicine Doctoral Dissertation Series
Abstract
Human papillomavirus type 16 (HPV16) is the most common high-risk type and is
associated with more than 50% cervical cancer cases as well as a number of head
and neck cancer cases. The life cycle of HPV16 is strictly linked to cell
differentiation in the squamous epithelium with expression of the HPV16 early
genes. The oncoproteins E6 and E7 are essential for preventing apoptosis and
inducing proliferation of HPV16 infected cells. As cell differentiation proceeds,
viral protein E2 suppresses early promoter p97 and results in downregulation of
E6/E7 proteins and activation of HPV16 late gene expression. It is not surprising
that the HPV16 E2 gene is inactivated in many HPV16-driven cancer... (More)
Human papillomavirus type 16 (HPV16) is the most common high-risk type and is
associated with more than 50% cervical cancer cases as well as a number of head
and neck cancer cases. The life cycle of HPV16 is strictly linked to cell
differentiation in the squamous epithelium with expression of the HPV16 early
genes. The oncoproteins E6 and E7 are essential for preventing apoptosis and
inducing proliferation of HPV16 infected cells. As cell differentiation proceeds,
viral protein E2 suppresses early promoter p97 and results in downregulation of
E6/E7 proteins and activation of HPV16 late gene expression. It is not surprising
that the HPV16 E2 gene is inactivated in many HPV16-driven cancer cells.
Therefore, the study of HPV16 early gene regulation especially regulation of E1,
E2, E6 and E7 expression is important to understand HPV16 infection. These
studies may contribute to development of antiviral drugs to HPV16.
The HPV16 E6 protein is translated from unspliced mRNAs, whereas the E7 protein
is translated from the mRNAs spliced from HPV16 5’-splice site SD226 to 3’-splice
site SA409. The balanced expression of unspliced and spliced mRNAs is important
to produce the E6 and E7 proteins that affect the HPV16 life cycle progression and
pathogenesis. We have investigated the regulation of splicing from SD226 to
SA409. We determined that cellular splicing factors hnRNP A1 and hnRNP A2B1
function as inhibitors of HPV16 E7 expression. Both hnRNP A1 and hnRNP A2
inhibited splicing to SA409 through direct and specific interaction with a C-less
RNA element located between HPV16 nucleotide position 594 and 604. Even
though inhibition SA409 exerted by hnRNP A1 and hnRNP A2 had different effects.
Overexpression of hnRNP A1 inhibited SA409 and resulted in enhanced expression
of unspliced E6 mRNAs at the expense of the E7 mRNAs. Overexpression of
hnRNP A2 inhibited SA409 but resulted in alternative splicing to 3’-splice site
SA742. The splice site SA742 is used for the production of the HPV16 E6^E7, E1,
and E4 mRNAs.
Since HPV16 E2 is a transcriptional regulator indirectly downregulates HPV16 E6
and E7 expression via suppressing of HPV16 early promoter p97. HPV16 produces
various E2 mRNAs that initiate at either early promoter p97 or late promoter p670
and several splice sites are used. Our study demonstrated that the most efficiently
translated E2 mRNA initiates at late promoter p670 spliced from 5’-splice site
SD880 to 3’-splice site SA2709. An alternative 3’-splice site named SA2582 and
located upstream of the E2 ATG could also produce E2 mRNAs, but it is less
efficiently to be translated into E2 protein. These results suggest that a splicing
enhancer may be located downstream of SA2709 to regulate E2 mRNA expression.
To further understand how HPV16 regulates 3’-splice site SA2709, we introduced
a number of deletions and mutations to HPV16 subgenomic plasmids. We identified
a 19-nucleotide RNA element consisting of three “AC(A/G)AGG” repeats located
downstream of SA2709.This RNA element acted as a splicing enhancer. This
splicing enhancer sequence interacted with cellular RNA binding protein hnRNP G
to enhance splicing to SA2709 and interacted with hnRNP A1 and A2B1 to inhibit
splicing to SA2709. Deletion of the splicing enhancer reduced mRNA splicing to
SA2709, and redirected splicing to the upstream 3’-splice site SA2582. The later
mRNA has poor ability to produce E2 protein, but it may produce a small protein
called E1C. We also wish to establish a bio-assay that can monitor expression of the
various E1 mRNAs and the alternatively spliced E2 mRNAs using either SA2582
or SA2709. We constructed a number of subgenomic HPV16 plasmids containing
green fluorescent protein, secreted luciferase, neomycin resistance gene, or
thymidine kinase reporter genes. These reporter plasmids produce either E1-fusion
proteins or E2-fusion proteins. We observed that the balance between unspliced E1
mRNAs and spliced E2 mRNAs was affected by overexpression of RNA binding
proteins SRp30c, Tra2b, hnRNP A1, and hnRNP G. However, further study is
required to optimize these reporter plasmids for quantitative analysis of HPV16 E1
and E2 mRNA splicing. This bioassay could be used to enhance our understanding
of HPV16 gene regulation and to identify small molecules that interfere with
HPV16 mRNA splicing. Such substances could potentially be used as antiviral
drugs to HPV16 infections and HPV16-driven cancers.
(Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Director and Professor of Molecular Biomedicine Kurg, Reet, Institute of Technology University of Tartu, Estonia
organization
publishing date
type
Thesis
publication status
published
subject
keywords
HPV16, splicing, mRNA, hnRNP A1, hnRNP G, E6, E7, E1, E2
in
Lund University, Faculty of Medicine Doctoral Dissertation Series
issue
2021:27
pages
90 pages
publisher
Lund University, Faculty of Medicine
defense location
Dora Jacobsohn, BMC D15, Klinikgatan 32 i Lund. Join by Zoom: https://lu-se.zoom.us/j/69985617527
defense date
2021-03-25 09:00:00
ISSN
1652-8220
ISBN
978-91-8021-033-1
language
English
LU publication?
yes
id
3e53811e-ab23-4e9f-be32-19ea6570b8a9
date added to LUP
2021-03-01 15:02:20
date last changed
2021-03-18 12:37:02
@phdthesis{3e53811e-ab23-4e9f-be32-19ea6570b8a9,
  abstract     = {{Human papillomavirus type 16 (HPV16) is the most common high-risk type and is<br/>associated with more than 50% cervical cancer cases as well as a number of head<br/>and neck cancer cases. The life cycle of HPV16 is strictly linked to cell<br/>differentiation in the squamous epithelium with expression of the HPV16 early<br/>genes. The oncoproteins E6 and E7 are essential for preventing apoptosis and<br/>inducing proliferation of HPV16 infected cells. As cell differentiation proceeds,<br/>viral protein E2 suppresses early promoter p97 and results in downregulation of<br/>E6/E7 proteins and activation of HPV16 late gene expression. It is not surprising<br/>that the HPV16 E2 gene is inactivated in many HPV16-driven cancer cells.<br/>Therefore, the study of HPV16 early gene regulation especially regulation of E1,<br/>E2, E6 and E7 expression is important to understand HPV16 infection. These<br/>studies may contribute to development of antiviral drugs to HPV16.<br/>The HPV16 E6 protein is translated from unspliced mRNAs, whereas the E7 protein<br/>is translated from the mRNAs spliced from HPV16 5’-splice site SD226 to 3’-splice<br/>site SA409. The balanced expression of unspliced and spliced mRNAs is important<br/>to produce the E6 and E7 proteins that affect the HPV16 life cycle progression and<br/>pathogenesis. We have investigated the regulation of splicing from SD226 to<br/>SA409. We determined that cellular splicing factors hnRNP A1 and hnRNP A2B1<br/>function as inhibitors of HPV16 E7 expression. Both hnRNP A1 and hnRNP A2<br/>inhibited splicing to SA409 through direct and specific interaction with a C-less<br/>RNA element located between HPV16 nucleotide position 594 and 604. Even<br/>though inhibition SA409 exerted by hnRNP A1 and hnRNP A2 had different effects.<br/>Overexpression of hnRNP A1 inhibited SA409 and resulted in enhanced expression<br/>of unspliced E6 mRNAs at the expense of the E7 mRNAs. Overexpression of<br/>hnRNP A2 inhibited SA409 but resulted in alternative splicing to 3’-splice site<br/>SA742. The splice site SA742 is used for the production of the HPV16 E6^E7, E1,<br/>and E4 mRNAs.<br/>Since HPV16 E2 is a transcriptional regulator indirectly downregulates HPV16 E6<br/>and E7 expression via suppressing of HPV16 early promoter p97. HPV16 produces<br/>various E2 mRNAs that initiate at either early promoter p97 or late promoter p670<br/>and several splice sites are used. Our study demonstrated that the most efficiently<br/>translated E2 mRNA initiates at late promoter p670 spliced from 5’-splice site<br/>SD880 to 3’-splice site SA2709. An alternative 3’-splice site named SA2582 and<br/>located upstream of the E2 ATG could also produce E2 mRNAs, but it is less<br/>efficiently to be translated into E2 protein. These results suggest that a splicing<br/>enhancer may be located downstream of SA2709 to regulate E2 mRNA expression.<br/>To further understand how HPV16 regulates 3’-splice site SA2709, we introduced<br/>a number of deletions and mutations to HPV16 subgenomic plasmids. We identified<br/>a 19-nucleotide RNA element consisting of three “AC(A/G)AGG” repeats located<br/>downstream of SA2709.This RNA element acted as a splicing enhancer. This<br/>splicing enhancer sequence interacted with cellular RNA binding protein hnRNP G<br/>to enhance splicing to SA2709 and interacted with hnRNP A1 and A2B1 to inhibit<br/>splicing to SA2709. Deletion of the splicing enhancer reduced mRNA splicing to<br/>SA2709, and redirected splicing to the upstream 3’-splice site SA2582. The later<br/>mRNA has poor ability to produce E2 protein, but it may produce a small protein<br/>called E1C. We also wish to establish a bio-assay that can monitor expression of the<br/>various E1 mRNAs and the alternatively spliced E2 mRNAs using either SA2582<br/>or SA2709. We constructed a number of subgenomic HPV16 plasmids containing<br/>green fluorescent protein, secreted luciferase, neomycin resistance gene, or<br/>thymidine kinase reporter genes. These reporter plasmids produce either E1-fusion<br/>proteins or E2-fusion proteins. We observed that the balance between unspliced E1<br/>mRNAs and spliced E2 mRNAs was affected by overexpression of RNA binding<br/>proteins SRp30c, Tra2b, hnRNP A1, and hnRNP G. However, further study is<br/>required to optimize these reporter plasmids for quantitative analysis of HPV16 E1<br/>and E2 mRNA splicing. This bioassay could be used to enhance our understanding<br/>of HPV16 gene regulation and to identify small molecules that interfere with<br/>HPV16 mRNA splicing. Such substances could potentially be used as antiviral<br/>drugs to HPV16 infections and HPV16-driven cancers.<br/>}},
  author       = {{Zheng, Yunji}},
  isbn         = {{978-91-8021-033-1}},
  issn         = {{1652-8220}},
  keywords     = {{HPV16, splicing, mRNA, hnRNP A1, hnRNP G, E6, E7, E1, E2}},
  language     = {{eng}},
  number       = {{2021:27}},
  publisher    = {{Lund University, Faculty of Medicine}},
  school       = {{Lund University}},
  series       = {{Lund University, Faculty of Medicine Doctoral Dissertation Series}},
  title        = {{Regulation of HPV16 Early Gene Expression}},
  url          = {{https://lup.lub.lu.se/search/files/94627234/Yunji_Zheng_web.pdf}},
  year         = {{2021}},
}